Cholangiocarcinoma is a highly malignant neoplasm of the biliary tract and its incidence and mortality is rising. Early diagnosis is difficult and currently there is no effective chemoprevention or treatment. Our long-term goal is to define the cellular mechanisms responsible for tumor development and progression in biliary epithelia and to develop new therapies for cholangiocarcinoma. During the previous grant period, we have provided data supporting the important role of cytosolic phospholipase A21 (cPLA21) and cyclooxygenase-2 (COX-2)-derived prostaglandin (PG) signaling in cholangiocarcinoma growth. We have shown that EP1 is a key prostaglandin receptor that importantly contributes to cPLA21/COX-2-induced cholangiocarcinogenesis through transactivation of epidermal growth factor receptor (EGFR). In the current continuation proposal, we hypothesize that transactivation of EGFR by EP1 is crucial for activation of 2-catenin and inducible nitric oxide synthase (iNOS) and that the interactions of these key molecules perpetuate bile duct inflammation and drive carcinogenesis. Consequently, we postulate that simultaneous inhibition of EP1 and EGFR may synergistically disrupt the key steps in cholangiocarcinogenesis and provide effective chemoprevention and treatment. The specific aims of this ongoing investigation are: (1) To evaluate our hypothesis that S-nitrosylation of cPLA21 and COX-2 protein is a novel mechanism by which iNOS regulates cholangiocarcinogenesis; (2) To examine the effect and mechanism for cPLA21/COX-2/PGE2-induced 2-catenin activation in human cholangiocarcinoma cells; and (3) To examine the role of EP1-mediated EGFR transactivation in iNOS expression and to evaluate the effect of concomitantly inhibiting EP1 and EGFR on cholangiocarcinoma growth. Complementary approaches of cultured cholangiocarcinoma cells and animal models will be employed. The proposed research is highly significant since it will investigate important signaling pathways during cholangiocarcinogenesis and evaluate novel targeted therapy for chemoprevention and treatment. PUBLIC HEALTH RELEVANCE Cholangiocarcinoma is a highly malignant epithelial neoplasm arising within the biliary tract and its incidence and mortality is rising. Recent studies in our lab demonstrate an important role of cytosolic phospholipase A21 (cPLA21) and cyclooxygenase-2 (COX-2)-derived prostaglandin (PG) signaling in cholangiocarcinoma growth. We have shown that inhibiting COX-2 prevents the growth of cholangiocarcinoma cells in culture and in SCID mice. However, in light of the increased cardiovascular side effect associated with some COX-2 inhibitors, it is imperative to develop alternative chemopreventive strategy that simultaneously targets PG and other related key signaling pathways in cholangiocarcinogenesis, which is expected to provide synergistic anti-tumor effect with lesser side effect. Our ongoing investigation will elucidate the interplays between the cPLA21 and COX-2-derived PG signaling and other growth-regulatory pathways such as EGFR, iNOS and 2-catenin. Further knowledge on these aspects will help develop more effective therapeutic strategy. Moreover, the effect of simultaneous inhibition of EP1 and EGFR on cholangiocarcinoma growth will be examined by using cultured tumor cells and experimental animal models. The proposed research will define the molecular mechanisms responsible for cholangiocarcinoma development and progression and provide important therapeutic implications for the chemoprevention and treatment of this devastating malignancy.